The synthesis of peptides by conventional stepwise solid phase methodologies is limited by low yields when synthesizing long peptides. (Merrifield et al. J. Am. Chem. Soc. 1963, 85, 2149-2154; Kent et al. Ann. Rev. Biochem. 1988, 57, 957-989.) To overcome this limitation, smaller synthetic peptides may be joined to one another by chemical ligation to produce longer peptide products.
A method for chemically ligating peptides was disclosed by Schnolzer et al.. (Schnolzer et al., Science 1992, 256, 221-225; Rose et al. J. Am. Chem. Soc. 1994, 116, 30-34; Liu et al. Proc. Natl. Acad. Sci. USA 1994, 91, 6584-6588). The methodology disclosed by Schnolzer involves a chemoselective reaction of unprotected peptide segments to give a product with an unnatural backbone structure at the site of ligation. This methodology makes possible the synthesis of peptides of greater size than attainable by standard methods of peptide synthesis. (Canne et al. J. Am. Chem. Soc. 1995, 117, 2998-3007; Baca et al. J. Am. Chem. Soc. 1995, 117, 1881-1887; Williams et al. J. Am. Chem. Soc. 1994, 116, 10797-10798). This methedology also makes possible the synthesis of peptides of unusual structure and topology. (Dawson et al. J. Am. Chem. Soc. 1993, 115, 7263-7266; Rose et al. J. Am. Chem. Soc. 1994, 116, 30-34; Muir et al. Biochemistry 1994, 33, 7701-7708; Canne et al. J. Am. Chem. Soc. 1995, 117, 2998-3007). The combined use of conventional stepwise solid phase peptide synthesis together with chemical ligation enables chemists to routinely make unprotected peptides of up to 60 amino acid residues in good yield and purity. (Schnolzer et al. Int. J. Pept. Protein Res. 1992, 40, 180-193). The combination of these two methodologies may also be employed to achieve a total chemical synthesis of proteins.
Another chemical ligation technique has been reported for the preparation of proteins having a native backbone structure (Dawson et al. Science 1994, 266, 776-779). This mode of chemical ligation is termed "native ligation." In this technique, an unprotected synthetic peptide bearing a C-terminal .alpha.-thioester is reacted in a chemoselective manner with an unprotected peptide containing an N-terminal Cys residue. Thiol exchange reaction yields an intial thioester-linked intermediate which spontaneously rearranges to give a native amide bond at the ligation site joining the two peptide segments, in the process regenerating the Cys side chain thiol. This version of native ligation uses chemistry first described by Wieland for reacting amino acids. (Wieland et al. Liebigs Ann. Chem. 1953, 583, 129-149.) As originally described, native ligation is restricted to joining peptide segments at an X-Cys bond. (Dawson et al. Science 1994, 266, 776-779.)
What is needed is a general method for joining a C-terminal .alpha.-thioester peptide segment to an N-terminal amino acid peptide segment, wherein the N-terminal amino acid peptide segment need not have an N-terminal cysteine.